Antiretroviral therapy with a combination of RT and PR inhibitors has had a profound effect upon the management of HIV-1 infection. However, under drug pressure, the virus is able to evolve into forms that are resistant to the available drug regimens. Thus, drug resistance has become the most significant challenge to AIDS therapy. We have been studying a pediatric population undergoing a clinical trial with protease inhibitors in combination with RT inhibitors. We have discovered that evolution of the protease sequence is accompanied by evolution of the sequence of the cleavage sites within Gag/Pol, thus affecting the efficiency of processing. Furthermore, the evolution of protease is accompanied by alterations in cleavage specificity. We hypothesize that the Gag/Pol cleavage sites and PR form a functional unit in which sequence evolution may be co- dependent. In our renewal period, we will exploit these discoveries to gain additional understanding of the mechanisms of Gag/Pol processing. To this end, we will pursue the following specific aims: Specific Aim I will analyze natural Gag/Pol alleles for processing phenotype in a bacterial expression developed in the current period of support. We will also map the determinants by preparation of chimeric gag/pol constructs and analyze replication of recombinant virus with natural or chimeric gag/pol regions. Specific Aim 2 will study the growth of recombinant virus in the presence of anti-protease drugs in culture. In addition, new protease alleles will be subcloned and expressed for analysis by studies of substrate specificity using a combinatorial library of substrates, and by analysis of the binding of inhibitors. Specific Aim 3 will study the effects of changes in the sequence of Gag processing sites on the efficiency of processing. We will prepare peptides representing products of Gag/Pol processing and test their ability to inhibit the enzyme. We will also conduct structural analyses of fusions of Gag/Pol proteins with an inactive form of HIV PR in order to determine the role of structural organization in the processing events.